Structure-Guided Identification of C3d Residues Essential for Its Binding to Complement Receptor 2 (CD21)

Abstract

A vital role for complement in adaptive humoral immunity is now beyond dispute. The crucial interaction is that between B cell and follicular dendritic cell-resident complement receptor 2 (CR2, CD21) and its Ag-associated ligands iC3b and C3dg, where the latter have been deposited as a result of classical pathway activation. Despite the obvious importance of this interaction, the location of a CR2 binding site within C3d, a proteolytic limit fragment of C3dg retaining CR2 binding activity, has not been firmly established. The recently determined x-ray structure of human C3d suggested a candidate site that was remote from the site of covalent attachment to Ag and consisted of an acidic residue-lined depression, which accordingly displays a significant electronegative surface potential. These attributes were consistent with the known ionic strength dependence of the CR2-C3d interaction and with the fact that a significant electropositive surface was apparent in a modeled structure of the C3d-binding domains of CR2. Therefore, we have performed an alanine scan of all of the residues within and immediately adjacent to the acidic pocket in C3d. By testing the mutant iC3b molecules for their ability to bind CR2, we have identified two separate clusters of residues on opposite sides of the acidic pocket, specifically E37/E39 and E160/D163/I164/E166, as being important CR2-contacting residues in C3d. Within the second cluster even single mutations cause near total loss of CR2 binding activity. Consistent with the proposed oppositely charged nature of the interface, we have also found that removal of a positive charge immediately adjacent to the acidic pocket (mutant K162A) results in a 2-fold enhancement in CR2 binding activity.

Mapping of the C3d Ligand Binding Site on Complement Receptor 2 (CR2/CD21) Using Nuclear Magnetic Resonance and Chemical Shift Analysis

“…In contrast to the emerging consensus regarding the CR2 side of the CR2-C3d interface, it is still uncertain where CR2 binds C3d (33,36,55). Our data do not directly address this question but are consistent with the co-crystal structure determination showing the SCR2-C3d interaction.…”

The Electrostatic Nature of C3d-Complement Receptor 2 Association

“…It has been recognized in a number of studies that C3d-CR2 association is pH-and ionic strength-dependent (20,(33)(34)(35)(36). Similar pH and ionic strength dependence was also observed in the homologous interaction between C4b and C4-binding protein (37,38).…”

“…The structure of free C3d became first available and revealed the presence of an acidic pocket that was thought to be a candidate for the association site with CR2 (39). Subsequent mutagenesis studies within the acidic pocket of C3d in iC3b demonstrated either loss or gain of binding ability to CR2(SCR1-15)-bearing Raji cells and supported this hypothesis (35). However, the structure of the C3d-CR2(SCR1-2) complex became available later, showing that the association site was not at, or close to, the acidic pocket (35).…”

Structural insights into the central complement component C3

“…This provides a link between the innate and the adaptive immune system (Carroll, 2004). Multiple studies have been performed on the interaction of CR2 with iC3b and C3dg (Clemenza and Isenman, 2000;Diefenbach and Isenman, 1995;Esparza et al, 1991;Kalli et al, 1991;Lambris et al, 1985;Lowell et al, 1989;Molina et al, 1995;Morikis and Lambris, 2004;Sarrias et al, 2001) (Fig. 2).…”